1. Technical Field
The present invention is related to the development of monoclonal antibodies against the ras p21 gene product and the use of genetically engineered deletion mutants to localize epitopes recognized by the antibodies. More particularly, the present invention is related to the use of monoclonals to dissect structural and functional properties of the oncogenic ras p21 protein and to detect ras p21 proteins in body tissue or body fluid as an indicator of the presence of malignant condition.
2. State of the Art
A small set of eukaryotic genes, termed proto-oncogenes, can be activated as oncogenes by a variety of mechanisms in naturally occurring tumor cells. These genes were initially detected as the transforming genes of retroviruses. Recent evidence has implicated their activation as oncogenes in as many as 10-30% of human malignancies by mechanisms involving point mutations at one of two major sites in their coding sequence (Capon et al., Nature 304: 507-513, 1983; Kraus et al., Proc. Natl. Acad. Sci. USA 81:5384-5388, 1984). The structural and functional characterization of ras genes has been significantly aided by expression of their p21 product at high levels in E. coli. Purification from bacterial extracts of normal and transforming p21s has also been successfully achieved (Lacal et al., Proc. Natl. Acad. Sci. USA 81:5305-5309, 1984; Stein et al., J. Virol. 50:343-351, 1984). Moreover, microinjection of low concentrations of the activated p21 product, or of much higher concentrations of normal p21, has been shown to induce morphological alterations as well as the induction of DNA synthesis in quiescent cells (Feramisco et al., Cell 38:109-117, 1984; Stacey and Kung, Nature 310:508-511, 1984).
In view of the importance of ras proto-oncogenes in the malignant process, there have been intensive investigations of the structure and function of p21 proteins. Biochemical activities associated with p21 proteins include GTP binding, GTP-dependent autophosphorylation and GTPase activities. Moreover, findings of some homology between ras proteins and known G proteins such as elongation factors and the .alpha. subunit of transducin suggest that the functions of p21 proteins are related to their ability to bind and hydrolyze GTP.
The ability of monoclonal antibodies to specifically recognize ras p21 proteins has provided essential tools for characterizing the processing, subcellular localization, and biochemical properties of the p21 molecule (Ulsh et al., Mol. Cell. Biol. 4:1647-1652, 1984).
To date, only a limited number of monoclonal antibodies have been generated against p21 (Furth et al., J. Virol 43:294-304, 1982) and their recognition sites on the p21 molecule have only been mapped in a very few cases.